This study describes the purification of Bixa orellana L. leaf lectin (BoLL) and the evaluation of its antimicrobial activity and in vivo toxicity. BoLL was isolated from saline extract of leaves through protein precipitation with ammonium sulfate (60% saturation) followed by ion exchange (CM-Sephadex) and size exclusion (Sephadex G-75) chromatographies. The lectin was characterized by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE), stability towards heating and pH variation, as well as influence of ions on its hemagglutinating activity (HA). HA inhibition assay by different carbohydrates and glycoproteins was also performed. The antimicrobial activity of BoLL was investigated against pathogens for humans and plants. In vivo acute toxicity in mice was assessed using a dose of 100 mg/kg per os or i.p. BoLL was isolated with high specific HA (purification fold: 53.89) and showed a single 19-kDa polypeptide band in SDS-PAGE. BoLL SHA increased in the presence of 20 mM Mg2+ ions, was resistant to heating at 100 °C, and stable at acidic pH range. HA inhibition was detected especially by glycoproteins, but also by monosaccharides. BoLL showed bacteriostatic and bactericidal effects against Escherichia coli minimum inhibitory (MIC) and bactericidal (MBC) concentrations of 400 and 800 μg/mL, respectively and Staphylococcus aureus (MIC and MBC: 400 μg/mL), as well as bacteriostatic effect against Bacillus megaterium and Micrococcus luteus (MIC: 18.2 and 800 μg/mL, respectively). No changes were observed in hematological, biochemical, and behavioral parameters in the acute toxicity test. However, animals treated with BoLL at 100 mg/kg per os or i.p. showed histopathological alterations in the liver, spleen, and kidneys. In conclusion, our data add new biotechnological value for B. orellana leaves, since they contain a thermostable lectin with antibacterial activity, which stimulate future studies aiming at its biomedical application. However, caution should be considered in future studies due to some signs of toxicity in mice. •A protocol for purification of a lectin from Bixa orellana leaf (BoLL) was defined.•BoLL showed a single 19-kDa polypeptide band in SDS-PAGE.•Hemagglutinating activity of BoLL increased in the presence of Mg2+ ions, was resistant to heating at 100 °C, and stable at acidic pH range.•BoLL showed bacteriostatic and bactericidal effects•BoLL (100 mg/kg per os or i.p.) did not cause the death of Swiss mice.